An understanding of how and why a protein folds into its native conformation is of major biological, medical and technical importance. This proposal is addressed to the question of how proteins fold, and factors which determine a particular folding pathway and hence native structure. The long-term goals are to learn the "rules" by which the amino acid sequence of a polypeptide guides the folding path to the native state. Such information should facilitate the prediction of three-dimensional structure based on amino acid or gene sequence. The approach chosen to accomplish this involves the following features: (1) specific environment-sensitive signals of probes in different regions of the molecule to aid in determining the order in which the protein folds and unfolds, i.e. to map out the folding pathway; (2) the use of site-specific mutants to facilitate the formation of protein with environment-sensitive probes in unique and selected sites, and (3) to probe the role of individual amino acids on the folding: (4) the use of subzero temperatures and aqueous-methanol cryosolvents to permit the stabilization and characterization of partially-folded intermediates in both unfolding and refolding, in addition to rapid kinetic techniques with aqueous solutions. The proposal is composed of the following components: (1) studies on the kinetics of folding of various mutant staphylococcal nucleases (SNase) in aqueous solution, (2) generation of SNase labeled in selected regions of the molecule by combining site- directed mutagenesis with chemical modification using environment-sensitive reporter groups, (3) folding kinetics experiments on SNase (wild-type and mutants), monitoring the process with environment-sensitive probes both at subzero temperatures and in aqueous solution using rapid-reaction methods, (4) characterization of selected, site-specific, mutants of staphylococcal nuclease, especially with respect to the kinetics of folding. The mutants to be examined are ones in which regions of secondary structure have been destabilized or in which a buried hydrophobic residue has been replaced by a more polar one. A major object is to determine the role of meta-stable regions of secondary structure in folding. Comparison of transients in the folding of mutants with that of the wild-type will permit inferences about the role of the mutated residue in the folding process.